1. Field of the Invention
The present invention relates to global regulators of bacterial pathogenic genes, and their use to confer disease resistance.
2. Description of the Related Art
A bibliography follows at the end of the Detailed Description of the Invention. The listed references are all incorporated herein by reference.
Cell-to-cell communication via small signal molecules is not only of vital importance to multi-celled living organisms such as animals and plants, it also plays important roles in the functional co-ordination among family members of single-celled organisms like bacteria. Rapid progress over the last few years has clearly established that N-acyl-homoserine lactones, known as autoinducers (AIs), are widely conserved signal molecules in Gram-negative bacteria. AIs were first found in marine bacteria Vibrio species in regulation of bioluminescence (Eberhard, et al., 1981; Cao and Meighen, 1989). In recent years, AIs have been identified in a wide range of Gram-negative bacteria. It has been found that AIs are involved in the regulation of a range of biological functions, including Ti plasmid conjugal transfer in Agrobacterium tumefaciens (Zhang, et al., 1993), induction of virulence genes in Erwinia carotovora, Pseudomonas aeruginosa, Erwinia stewartii, Xenorhabdus nematophilus, Erwinia chrysanthemi, Pseudomonas solanacerum, and Xanthomonas campestris (Jones, et al., 1993; Passador, et al., 1993; Pirhonen, et al., 1993; Pearson, et al., 1994; Beck von Bodman and Farrand, 1995; Barber, et al., 1997; Clough, et al., 1997; Costa and Loper, 1997; Dunphy, et al., 1997; Nasser, et al., 1998), regulation of antibiotics production in Pseudomonas aureofaciens and Erwinia carotovora (Pierson, et al., 1994; Costa and Loper, 1997), regulation of swarming motility in Serratia liquifaciens (Eberl, et al., 1996), and biofilm formation in Pseudomonas fluorescens and P. aeruginosa (Allison, et al., 1998; Davies, et al., 1998). Many more bacterial species are known to produce AIs but the biological functions related have not been established yet (Bassler, et al., 1997; Dumenyo, et al., 1998; Cha, et al., 1998).
Different bacterial species could produce different AIs. All AI derivatives share identical homoserine lactone moieties but can differ in the length and the structure of their acyl groups. The key components in AI-mediated gene regulation systems are LuxI and LuxR type proteins. It has been established now that LuxI-type protein serves as an autoinducer synthase that utilizes acyl-ACPs and AdoMet (S-adenosylmethionine) as substrates (More, et al., 1996; Schaefer, et al., 1996). LuxR-type protein is proposed to be both a receptor for AIs and a AI-dependent transcriptional regulator that binds DNA immediately upstream of the lux promoter (Meighen, 1994; Sitnikov, et al., 1995). A 20-nucleotide inverted repeat has been identified which is centered 44 nucleotides upstream of the transcription start site of the luminescence operon. This sequence called lux box is required for transcriptional activation by LuxR and is probably the LuxR binding site (Fuqua, et al., 1994). Similar 18-bp tra boxes are found upstream of at least three TraR-regulated promoters, and disruption of these elements abolishes transcriptional activation by TraR (Fuqua and Winans, 1996a).
LuxR-type proteins appear to be composed of two modules (Choi and Greenberg, 1991; Hanzelka and Greenberg, 1995). Their carboxyl terminal regions contain a conserved short sequence of 19-amino acid, putative probe-type helix-turn-helix motif, predicted to be involved in binding to target promoters. A general mechanism of activation has been proposed by which the N-terminal domain of LuxR-type protein acts negatively to prevent an interaction between its C-terminal domain and the target DNA binding sites. This inhibition can be relieved by the action of an autoinducer ligand. A strong piece of evidence is that deletion of the N-terminal domain of LuxR results in constitutively active alleles of luxR, whereas larger deletions that remove part of the predicted DNA binding domain abolish transcriptional activation (Choi and Greenberg, 1991). However, other members might use different mechanisms. Recent genetic studies indicate that EsaR and ExpR are likely to be repressors of their target genes rather than activators. Expression of the genes that are repressed by EsaR and ExpR is increased by autoinducers (Beck von Bodman and Farrand 1995; Throup, et al. 1995). It appears that binding of these proteins to their target sites in promoter region causes repression, therefore autoinducer ligands may act to reduce binding affinity.
Evidence that the autoinducer binding site resides in the amino terminal domain of the LuxR protein has been presented (Hanzelka and Greenberg, 1995). LuxR alleles that have mutated amino terminal region require higher level of this signal that does the wild type, indicating this region required for ligand interaction (Slock, et al., 1990; Shadel, et al., 1990). This region (aa 79-127) and a region within the DNA-binding domain (aa 180-230) show a higher degree of conservation among LuxR and its homologs (ca 50% identity) than other parts of these polypeptides. However, the proposed protein-ligand interaction between LuxR and autoinducer has not been proved yet. Analysis of merodiploid E. coli strains containing wild-type and mutant LuxR alleles suggested that LuxR functions as a homomultimer and that a region required for multimerization resides within amino acid residues 116 and 161 (Choi and Greenberg, 1992).